The primary objective of my proposed research is a precise biochemical elucidation of the structure and function of the molecular components that are involved in the selection translocation of certain classes of proteins across the membrane of the rough endoplasmic reticulum (RER). More specifically, I propose studies which comprise an in depth analysis of those components of the translation apparatus that are already identified (namely, the Signal Recognition Particle (SRP), a rebonucleoprotein, and the SRP receptor, an integral membrane protein of the RER), as well as attempt to identify and characterize further components of this protein translocation machinery. In the period covered by this proposal, the studies will concentrate on achieving a fuller molecular understanding of SRP and SRP's functional interactions with the SRP receptor protein, the ribosome and the signal sequences of the translocated proteins. Specifically, i) the structural features of the RNA-protein interactions in SRP will be precisely determined. ii) Specific antibodies raised against defined antigenic determinants on SRP, and cDNA probes complementary to specific regions of SRP-RNA will be used to localize different functional domains within SRP. iii) SRP will be dissected into its individual polypeptide and RNA constituents and reconstituted into a functional particle. The individual components and partially reconstituted SRP molecules will be used to elucidate the function of the particular polypeptide and RNA segments in SRP. iv) SRP and SRP receptor will be used as affinity reagents in attempts to identify, purify and characterize other components actively involved in the protein translocation machinery of the RER. Appropriate assays will be designed to elucidate the particular role of these components in the translocation process. Ultimately, all of the essential and modulatory components of the protein translocation machinery will be isolated and thoroughly characterized, such that the system can be functionally reconstituted from its parts into a phospholipid bilayer. Questions concerning the mechanism of the actual transmembrane movement of polypeptides will be experimentally addressed. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.